We describe methods for generating artificial transcription factors capable of up- or downregulating the expression of genes whose promoter regions contain the target DNA sequences. To accomplish this, we screened zinc fingers derived from sequences in the human genome and isolated 56 zinc fingers with diverse DNA-binding specificities. We used these zinc fingers as modular building blocks in the construction of novel, sequence-specific DNA-binding proteins. Fusion of these zinc-finger proteins with either a transcriptional activation or repression domain yielded potent transcriptional activators or repressors, respectively. These results show that the human genome encodes zinc fingers with diverse DNA-binding specificities and that these domains can be used to design sequence-specific DNA-binding proteins and artificial transcription factors.

Growth of Escherichia coli in the presence of glyphosate, an inhibitor of aromatic amino acid biosynthesis, has permitted the production of proton translocating ATPase that is specifically labelled with 5-fluorotryptophan. Five sets of tgF nuclear magnetic resonances are resolved. The use of glyphosphate should be of wide applicability in the preparation of proteins labelled in aromatic ammo acid residues for NMR studies.

Abnormal human hemoglobins (Hbs) with amino acid substitutions in the al132 interface have very high oxygen affinity and greatly reduced cooperativity in O2 binding compared to normal human Hb. In such abnormal Hbs with mutations at position 1899, the intersubunit hydrogen bonds between Asp-j399 and Tyr-a42 and between Asp-1399 and Asn-a97 are broken, thus destabi the deoxyquaternary structure of these Hbs. A molecular dynamics method has been used to design compensatory amino acid substitutions in these Hbs that can restore their allosteric properties. We have designed a compensatory mutation in a naturally occurring mutant Hb, Hb Kempsey (Asp-I399--Asn), and have produced it using ourEscherichia coli expression plasmid pHE2. We MATERIALS AND METHODSPlasmids, Strains, and Media. The Hb A expression plasmid pHE2 (15) containing synthetic a-and (-globin genes and the E. coli methionine aminopeptidase gene was used to produce mutant Hbs. Phagemid pTZ18U and E. coli JM109 were purchased from Bio-Rad and Promega, respectively. E. coli cells were grown in 2x YT medium (18) supplemented Abbreviations: Hb, hemoglobin; Hb A, human adult Hb; r, recombinant; MD, molecular dynamics; plo, partial pressure at 50%o oxygenation; nmax, Hill coefficient; DSS, 2,2-dimethyl-2-silapentane-5-sulfonate; IHP, inositol hexaphosphate.§To whom reprint requests should be addressed. 11547The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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According to the X-ray crystallographic results from human deoxyhemoglobin, beta 99Asp at the alpha 1 Beta 2 interface forms hydrogen bonds with alpha 42Tyr and alpha 97Asn. To clarify the structural and functional roles of the hydrogen bond between alpha 97Asn and beta 99Asp, we have engineered a recombinant hemoglobin in which alpha 97Asn is replaced by Ala, and have investigated its oxygen-binding properties, and have used proton nuclear magnetic resonance spectroscopy to determine the structural consequences of the mutation. Recombinant Hb (alpha 97Asn-->Ala) shows a milder alteration of functional properties compared to the severely impaired beta 99 mutants of the human abnormal hemoglobins. The addition of inositol hexaphosphate, an allosteric effector, causes recovery of the functional properties of recombinant Hb (alpha 97 Asn-->Ala) almost to the level of human normal adult hemoglobin without this allosteric effector. r Hb (alpha 97 Asn-->Ala) shows very similar tertiary structure around the heme pockets and quaternary structure in the alpha 1 beta 2 interface compared to those of human normal adult hemoglobin. The proton nuclear magnetic resonance spectrum of the deoxy form of this recombinant hemoglobin shows the existence of an altered hydrogen bond which is believed to be between alpha 42Tyr and beta 99Asp at the alpha 1 beta 2 interface. Thus, the present results suggest that the intersubunit hydrogen bond between alpha 97 Asn and beta 99Asp at the alpha 1 beta 2 interface is not as crucial as the one between alpha 42Tyr and beta 99Asp in the deoxy quaternary structure. Preliminary molecular dynamics simulations have been used to calculate the contributions of specific interactions of several amino acid residues in r Hb (alpha 97Asn-->Ala) to the free energy of cooperativity of this recombinant hemoglobin. The results of these calculations are consistent with the experimental results.